Regulation of neutrophil survival/apoptosis by Mcl-1.
Ontology highlight
ABSTRACT:
SUBMITTER: Milot E
PROVIDER: S-EPMC3217587 | biostudies-other | 2011
REPOSITORIES: biostudies-other
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Project description:Our previous study demonstrated that neutrophils play an important role in host defense against Coxiella burnetii infection in mice. In this study, avirulent strain C. burnetii Nine Mile phase II (NMII) was used to examine if C. burnetii can modulate mouse bone marrow-derived neutrophil apoptosis. The results indicated that NMII can inhibit neutrophil apoptosis. Western blotting demonstrated that caspase-3 cleavage was decreased in NMII-infected neutrophils, while phosphorylated mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase 1 (Erk1) were increased. Additionally, p38, Erk1/2, phosphoinositide 3-kinase (PI3K), or NF-?B inhibitors reduced the ability of NMII to inhibit neutrophil apoptosis. These results suggest that NMII-mediated inhibition of neutrophil apoptosis depends on its ability to activate neutrophil MAPK pathways. Antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) was significantly increased in NMII-infected neutrophils, and an Mcl-1 inhibitor significantly reduced the ability of NMII to inhibit neutrophil apoptosis. Mcl-1 protein stability was enhanced by phosphorylation at Thr-163 by Erk, and the protein levels were regulated by p38, Erk, PI3K, and NF-?B. Furthermore, the observation that a type IV secretion system (T4SS)-deficient dotA mutant showed a significantly reduced ability to inhibit neutrophil apoptosis compared to wild-type (WT) NMII suggests that T4SS-secreted factors may be involved in NMII-induced inhibition of neutrophil apoptosis. Collectively, these results demonstrate that NMII inhibits neutrophil apoptosis through inhibition of caspase-3 cleavage and activation of MAPK survival pathways with subsequent expression and stabilization of antiapoptotic protein Mcl-1, a process that may partially require the T4SS.
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Project description:Neutrophil apoptosis and subsequent nonphlogistic clearance by surrounding phagocytes are key to the successful resolution of neutrophilic inflammation, with dysregulated apoptosis reported in multiple human inflammatory diseases. Enhancing neutrophil apoptosis has proresolution and anti-inflammatory effects in preclinical models of inflammation. Here we investigate the ability of the flavones apigenin, luteolin, and wogonin to induce neutrophil apoptosis in vitro and resolve neutrophilic inflammation in vivo. Human neutrophil apoptosis was assessed morphologically and by flow cytometry following incubation with apigenin, luteolin, and wogonin. All three flavones induced time- and concentration-dependent neutrophil apoptosis (apigenin, EC=12.2 ?M; luteolin, EC=14.6 ?M; and wogonin, EC=28.9 ?M). Induction of apoptosis was caspase dependent, as it was blocked by the broad-spectrum caspase inhibitor Q-VD-OPh and was associated with both caspase-3 and caspase-9 activation. Flavone-induced apoptosis was preceded by down-regulation of the prosurvival protein Mcl-1, with proteasomal inhibition preventing flavone-induced Mcl-1 down-regulation and apoptosis. The flavones abrogated the survival effects of mediators that prolong neutrophil life span, including lipoteichoic acid, peptidoglycan, dexamethasone, and granulocyte-macrophage colony stimulating factor, by driving apoptosis. Furthermore, wogonin enhanced resolution of established neutrophilic inflammation in a zebrafish model of sterile tissue injury. Wogonin-induced resolution was dependent on apoptosis in vivo as it was blocked by caspase inhibition. Our data show that the flavones induce neutrophil apoptosis and have potential as neutrophil apoptosis-inducing anti-inflammatory, proresolution agents.
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Project description:Human tissue inflammation is terminated, at least in part, by the death of inflammatory neutrophils by apoptosis. The regulation of this process is therefore key to understanding and manipulating inflammation resolution. Previous data have suggested that the short-lived pro-survival Bcl-2 family protein, Mcl-1, is instrumental in determining neutrophil lifespan. However, Mcl-1 can be cleaved following caspase activity, and the possibility therefore remains that the observed fall in Mcl-1 levels is due to caspase activity downstream of caspase activation, rather than being a key event initiating apoptosis in human neutrophils.We demonstrate that apoptosis in highly purified neutrophils can be almost completely abrogated by caspase inhibition with the highly effective di-peptide caspase inhibitor, Q-VD.OPh, confirming the caspase dependence of neutrophil apoptosis. Effective caspase inhibition does not prevent the observed fall in Mcl-1 levels early in ultrapure neutrophil culture, suggesting that this fall in Mcl-1 levels is not a consequence of neutrophil apoptosis. However, at later timepoints, declines in Mcl-1 can be reversed with effective caspase inhibition, suggesting that Mcl-1 is both an upstream regulator and a downstream target of caspase activity in human neutrophils.
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Project description:Dengue is a global health concern, and the host-viral interactions that regulate disease severity are largely unknown. Detrimental effects of neutrophils in this disease have been reported, but the precise mechanisms and functional properties of dengue-activated neutrophils are not fully characterised. Here, we measured the effects of dengue virus serotype 3 (DV3) on neutrophil lifespan and functions. We show that DV3 extends neutrophil survival with a significant proportion of cells surviving for 72 h post-incubation. These effects on neutrophil survival were greater than those observed by adding GM-CSF and TNF-α alone, but these cytokines enhanced survival induced by the virus. Enhanced reactive oxygen species (ROS) generation was observed following incubation with DV3 activation and this ROS production was enhanced by co-incubation with priming agents. In addition, DV triggered the enhanced IL-8 expression by the majority of neutrophils and a low percentage of cells were activated to express MCP-1 (CCL2). A low number of neutrophils showed increased co-expression of the migratory markers, CCR7 and CXCR4 which could promote their migration towards lymph nodes. DV3 significantly upregulated the BCL-XL gene at 3, 12, and 24 h, and the Mcl-1 gene at 12 h, following treatment. We also show that DV3 induces the Mcl-1 protein stabilization similar to GM-CSF. This report sheds new light on the mechanisms by which neutrophils may contribute to the pathology of dengue disease via delayed apoptosis and generation of pro-inflammatory molecules, and raises the possibility that dengue-activated neutrophils may play a role in activating cells of adaptive immunity.
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Project description:During immune responses, neutrophils must integrate survival and death signals from multiple sources to regulate their lifespan. Signals that activate either the Bcl-2- or death receptor-regulated apoptosis pathways can provide powerful stimuli for neutrophils to undergo cell death, but whether they act cooperatively in parallel or directly cross-talk in neutrophils is not known. Previous studies suggested that Bcl-2 family proteins are not required for Fas-induced cell death in neutrophils, but did not examine whether they could modulate its rapid onset. By monitoring the rate of change in neutrophil viability associated with activation of the Fas-triggered death receptor pathway using real-time cell imaging, we show that the Bcl-2-related proteins Bid, Bax, and Bak accelerate neutrophil apoptosis but are not essential for cell death. Increased Bcl-2 or Mcl-1 expression prevents efficient induction of apoptosis by Fas stimulation indicating that the Bcl-2-regulated apoptosis pathway can directly interfere with Fas-triggered apoptosis. Fas has been shown to initiate NF?B activation and gene transcription in cell lines, however gene transcription is not altered in Fas-activated Bid(-/-) neutrophils, indicating that apoptosis occurs independently of gene transcription in neutrophils. The specification of kinetics of neutrophil apoptosis by Bid impacts on the magnitude of neutrophil IL-1? production, implicating a functional role for the Bcl-2-regulated pathway in controlling neutrophil responses to FasL. These data demonstrate that the intrinsic apoptosis pathway directly controls the kinetics of Fas-triggered apoptosis in neutrophils.
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Project description:MCL-1 (myeloid cell leukemia-1), a member of the BCL-2 family, has three splicing variants, antiapoptotic MCL-1L, proapoptotic MCL-1S, and MCL-1ES. We previously reported cloning MCL-1ES and characterizing it as an apoptotic molecule. Here, we investigated the molecular mechanism by which MCL-1ES promotes cell death. MCL-1ES was distinct from other proapoptotic BCL-2 members that induce apoptosis by promoting BAX or BAK oligomerization, leading to mitochondrial outer membrane permeabilization (MOMP), in that MCL-1ES promoted mitochondrial apoptosis independently of both BAX and BAK. Instead, MCL-1L was crucial for the apoptotic activity of MCL-1ES by facilitating its proper localization to the mitochondria. MCL-1ES did not interact with any BCL-2 family proteins except for MCL-1L, and antiapoptotic BCL-2 members failed to inhibit apoptosis induced by MCL-1ES. The BCL-2 homology 3 (BH3) domain of MCL-1ES was critical for both MCL-1ES association with MCL-1L and apoptotic activity. MCL-1ES formed mitochondrial oligomers, and this process was followed by MOMP and cytochrome c release in a MCL-1L-dependent manner. These findings indicate that MCL-1ES, as a distinct proapoptotic BCL-2 family protein, may be useful for intervening in diseases that involve uncontrolled MCL-1L.
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Project description:The development of selective inhibitors for discrete anti-apoptotic BCL-2 family proteins implicated in pathologic cell survival remains a formidable but pressing challenge. Such precisely tailored compounds would serve as molecular probes and targeted therapies to study and treat human diseases driven by specific anti-apoptotic blockades. In particular, MCL-1 has emerged as a major resistance factor in human cancer. By screening a library of stabilized alpha-helix of BCL-2 domains (SAHBs), we determined that the MCL-1 BH3 helix is itself a potent and exclusive MCL-1 inhibitor. X-ray crystallography and mutagenesis studies defined key binding and specificity determinants, including the capacity to harness the hydrocarbon staple to optimize affinity while preserving selectivity. MCL-1 SAHB directly targets MCL-1, neutralizes its inhibitory interaction with pro-apoptotic BAK and sensitizes cancer cells to caspase-dependent apoptosis. By leveraging nature's solution to ligand selectivity, we generated an MCL-1-specific agent that defines the structural and functional features of targeted MCL-1 inhibition.
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